TRAK2 interacts and colocalizes with Miro1-var4 on peroxisomes. (A) HEK cells were transfected with HA₂–Miro1-var4 or HA₂–Miro1-var1 with or without FLAG-TRAK2 as indicated at the top. At 24 h after transfection, cells were solubilized and subjected to immunoprecipitation with anti-FLAG antibody. Immunoprecipitates (IPs; lanes 5–8) and the input (10%; lanes 1–4) were analyzed by SDS-PAGE and immunoblotting with antibodies as indicated on the left. (B) HeLa cells were cotransfected for 24 h with HA₂–Miro1-var4 and FLAG-TRAK2 and immunostained with antibodies to FLAG (a and e; green), HA (b and f; red), and Pex14p (c and g; blue). Merged images were shown (d and h), and the boxed areas were fivefold magnified (e–h). Bars: (a–d) 10 µm; (e–h) 2 µm. Arrowheads indicate colocalization of FLAG-TRAK2 with HA₂–-Miro1-var4 in peroxisomes in the cell periphery region. (C) A PNS fraction (lane 2) from HEK cells transiently expressing HA₂-TRAK2 was centrifuged at 3,000 g and separated into supernatant (3Kg sup; lane 1) and pellet (3Kg pellet; lane 3) fractions as in Fig. 2 C. The 3Kg sup fraction was further centrifuged at 100,000 g to yield a cytosol-free pellet fraction (100Kg pellet; lane 4). The resulting 100Kg pellet was solubilized and subjected to immunoprecipitation with antisera to Miro1-ins1 (lane 7), HA (lane 9), and their respective preimmune sera (pre-imm.; lanes 6 and 8). Equal-volume aliquots of separated fractions (lanes 1–4) and the immunoprecipitates (lanes 6–9) together with the input of the 100Kg pellet (5%; lane 5) were analyzed by SDS-PAGE and immunoblotting with antibodies indicated on the left.